Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in the mouse uterus

Early gestational loss occurs in approximately 20% of all clinically recognized human pregnancies and is an important cause of morbidity. Either embryonic or maternal defects can cause loss, but a functioning and receptive uterine endometrium is crucial for embryo implantation. We report that the switch/sucrose nonfermentable (SWI/SNF) remodeling complex containing polybromo-1 (PBRM1) and Brahma-related gene 1 (BRG1) is essential for implantation of the embryonic blastocyst on the wall of the uterus in mice. Although preimplantation development is unaffected, conditional ablation of Pbrm1 in uterine stromal cells disrupts progesterone pathways and uterine receptivity. Heart and neural crest derivatives expressed 2 (Hand2) encodes a basic helix-loop-helix (bHLH) transcription factor required for embryo implantation. We identify an enhancer of the Hand2 gene in stromal cells that requires PBRM1 for epigenetic histone modifications/coactivator recruitment and looping with the promoter. In Pbrm1cKO mice, perturbation of chromatin assembly at the promoter and enhancer sites compromises Hand2 transcription, adversely affects fibroblast growth factor signaling pathways, prevents normal stromal-epithelial crosstalk, and disrupts embryo implantation. The mutant female mice are infertile and provide insight into potential causes of early pregnancy loss in humans.


Figure S4 .
Figure S4.Chromatin accessibility in Pbrm1-mutant primary uterine stromal cells.(A) ATAC-seq heatmap of D4 uterine stromal cells from WT and Pbrm1 deletion female mice.(B) Heatmap showing distribution of WT and Pbrm1 cKO ATAC-seq peak regions centered at transcriptional start site (TSS).(C-D) Genomic distribution of WT and Pbrm1 cKO peaks.(E) KEGG functional analysis for differential peaks in the ATAC-seq down regulated genes (dn) that overlap RNA-seq down regulated genes.(F) Clustered heat maps displaying PBRM1 and BRG1 normalized ChIC-seq peaks centered at the TSS in WT D4 uterine stromal cells.(G) Genomic distribution of PBRM1 and BRG1 ChIC peaks in WT primary uterine stromal cell on D4.

Figure S5 .
Figure S5.Comparable Hand2 transcription level and chromatin accessibility in Pbrm1 fl/fl and Pbrm1 cKO primary mice oviductal smooth muscle cells.Genome browser view of normalized RNA-seq signals and ATAC-seq tracks for Hand2 in Pbrm1 fl/fl and Pbrm1 cKO primary mice oviductal smooth muscle cell.Green rectangle, a newly identified uterine specific enhancer regulated by SWI/SNF complex.Blue rectangle, known branchial arch enhancer.Black rectangle, cardiac-specific enhancer.Red rectangle, promoter of Hand2 directly bound by the SWI/SNF complex.Rep 1, 2 and 3, three biological replicates.

Figure S6 .
Figure S6.Predicted binding motifs in the Hand2 gene locus and uterine specific enhancer knockout mice generated with CRISPR/Cas9.(A-B) Predicted binding motifs in the middle of putative uterine specific Hand2 enhancer region.Luciferase constructs with the putative Hand2 WT (A) and mutated motifs (B) enhancer region.The red highlights motifs 1-4.(C-D) The Hand2 heart and branchial arch specific enhancers did not have the PBRM1/BRG1 specific binding motifs.(E) Design of the putative uterine specific Hand2 enhancer region and DNA sequence at the enhancer locus of 2 knockout mouse lines.

Figure S7 .
Figure S7.Exogenous progesterone supplementation cannot improve embryo implantation in Pbrm1 mutant females.Implantation rate and representative morphology of uteri from Pbrm1 fl/fl and Pbrm1 cKO female mice treated with oil or P4.Number within the bar indicates the number of mice with implantation sites per total tested mice.

Figure S8 .
Figure S8.PBRM1, BRG1 and HAND2 expression in human uterine stromal cells.(A) Single cell RNA-seq documents co-expression of PBRM1, BRG1 and HAND2 in normal pregnant women uterine stromal cells (data from GSE194219).(B) Immunostaining of PBRM1, BRG1 and HAND2 demonstrates co-localization in the nuclei of human uterine stromal cells.(C) Confocal images of GFP documents transduction efficiency after lentivirus infection of human uterine stromal cells.Scale bar: 100 µm.(D) Immunoblot analysis of PBRM1 protein expression in human uterine stromal cells.β-actin, loading control.(E) Quantitative real-time PCR of HAND2 in PBRM1 knockdown of human uterine stromal cells.Data shown represent the mean ± SEM. *Ρ <0.05, independent-sample Student's t test.